The newly described collagen fragment proline-glycine-proline (PGP) drives chronic neutrophilic inflammatory responses and appears to play a role in COPD pathogenesis. Prolyl endopeptidase (PE) is a particularly important serine protease because it has been shown to catalyze the final step in a proteolytic cascade required to generate PGP from collagen. Consequently, PE would seem to represent a very attractive new target for future therapeutics related to COPD. However because recognition of the role of PE in chronic inflammation is so new, certain fundamental aspects of the regulation of this protease in the context of lung disease remains poorly understood. This revised proposal will fill in this void of knowledge by highlighting new evidence from the PI's laboratory demonstrating that PE is released from airway epithelial cells via exosomes. This application focuses on a unique feed-forward pathway of PE expression and PGP generation in cigarette smoke (CS) exposure and COPD with particular emphasis on endogenous CXCR ligand signaling. The aims in this proposal are thematically linked to each other (through high quality in vitro, in vivo models and ex vivo clinicl specimens) and are carefully crafted to improve our understanding of this pathway and its relevance to COPD lung disease. Specific aim 1a will examine the regulation and release of PE from primary airway epithelial cells by CXC ligands, highlighting the potential of PE release via exosomes, identifying the intracellular pathways involved in the release, and determining the impact of this release on ex vivo PGP generation from collagen. Specific aim 1b will determine the impact of cigarette smoke (CS) on the regulation of PE and PGP production, with specific targeting of CXC receptors and PE with novel therapeutic compounds. The second aim of this grant will focus on PE inhibition as a therapeutic approach in a chronic CS model of COPD. This will be examined utilizing a genetic deletion of PE (the PREP-/- mouse) to examine if there is amelioration of the observed inflammatory response and phenotype with chronic CS exposure. This model will also be examined in testing a new PE inhibitor S- 17092, currently in clinical trials for neurodegenerative disease, as a lead anti-inflammatory therapeutic in this model of COPD lung disease. The final aim will involve the collection of clinical biospecimens via bronchoscopy from patients with COPD to determine the presence of PE-rich exosomes and PGP peptides compared to individuals without lung disease. The successful completion of these aims will lead to an increased understanding of the regulation and release of PE in the extracellular environment and the downstream effects of its unfettered protease activity. In doing so, these studies may result in the development of a new biomarker and therapeutic target for COPD.